Identification and characterization of the VAX2 p.Leu139Arg variant: possible involvement of VAX2 in cone dystrophy

<p><b>Objective</b>: This study was undertaken with the objective to investigate the potential involvement of VAX2 in retinal degeneration.</p> <p><b>Methods</b>: A cohort of macular and cone dystrophy patients (<i>n</i> = 70) was screened for variant identification. Polymerase chain reaction (PCR) products were purified using ExoSAP-IT. Direct sequencing of PCR products was performed using BigDye 3.1 on the ABI 3730 DNA Analyzer and analyzed using DNASTAR software tool. Search for known variant was performed using the following platforms: 1000 Genomes Project, Ensembl, UCSC, ExAc, and dbSNP. The <i>VAX2</i> mutants were generated using the GeneArt® Site-Directed Mutagenesis kit. <i>In vitro</i> analysis was performed in hTERTRPE-1 (RPE-1) cell line. Cells were photographed using a Zeiss AXIOVERT S100 microscope. Images were analyzed using Photoshop CS4 software.</p> <p><b>Results</b>: Here, we report the identification of a heterozygous non-synonymous variant (c.416T>G; p.Leu139Arg) in one cone dystrophy proband. Functional characterization of this variant <i>in vitro</i> revealed an aberrant phenotype seen as protein mislocalization to cytoplasm/nucleus and aggregates undergoing degradation or forming aggresomes. The cellular phenotype suggests protein loss-of-function. Analysis of the <i>VAX2</i> p.Leu139Met, a variant present in the normal population, showed a phenotype similar to the wild-type, further supporting the hypothesis for the Leucine 139 to Arginine change to be damaging.</p> <p><b>Conclusions</b>: This study raises the interesting possibility for evaluating <i>VAX2</i> as a candidate gene for cone dystrophy.</p

<i>CNOT3</i> shows an opposite trend of expression with respect to that of <i>PRPF31</i> between the asymptomatic (AS) and affected (AF) individuals of the AD5 family.

<p>(A) <i>PRPF31</i> mRNA expression normalized to the housekeeping gene <i>GAPDH</i>. Error bars refer to the standard deviation of the mean for 5 independent experiments for each group. (B) <i>CNOT3</i> mRNA expression from the same 5 experiments used to generate <i>PRPF31</i> data. **, <i>p</i><0.01. (C) Linear regression analysis of <i>PRPF31</i> and <i>CNOT3</i> mRNA expression, which shows an inverse trend of the two genes in each cell line. Circles, asymptomatic subjects; triangles, affected individuals; open symbols, <i>CNOT3</i> expression; Filled symbols, <i>PRPF31</i> expression. Data having the same value for the x axis have been obtained from the same individual. (D) Quantification of CNOT3 protein abundance relative to β-actin from 3 independent SDS-PAGE gels, after simultaneous detection of the two proteins by quantitative LI-COR western blot.</p

Analysis of rs4806718 alleles in two unrelated pedigrees.

<p>(A) Family RP856/AD5. The individuals initially tested with NGS are marked with a star. The individuals marked with a triangle belong to a sibship pair, which was previously shown by McGee et al. to have the same isoallele haplotype but different phenotypes. (B) Family ADB1, a Bulgarian gypsy family carrying a heterozygous splice site mutation in <i>PRPF31</i> (NM_015629.3:c.527+1G>T, or IVS6+1G>T). In both pedigrees carriers of mutations are either in black (affected individuals) or in grey (asymptomatic individuals).</p

P26s4 interacts with TOPORS in yeast and in human cell lines.

<p><b>A)</b> TOPORS protein domain diagram. Black ovals denote known phosphorylation sites at serines 98 and 718 required for regulation of E3 ligase activities of TOPORS; dark red box (residues 103–141) indicates the RING domain; dark cross-hatched box (residues 530–777) represents the arginine- and serine-rich (SR/RS) domain; a SUMO1 acceptor site at Lys 560 with a covalently bound SUMO1 modification (perpendicular black box) is indicated within the SR/RS domain; blue boxes (residues 415–737 and 854–1045) indicate regions required for interaction with SUMO1; black box (residues 437–574) represents a fragment required for SUMOylation of TOPORS at Lys 560; the green box (aa: 871–917) represents a region required for minimal interaction with UBC9. The pink asterisk represents the <i>RP31</i> mutational hotspot. The horizontal red, blue and green bars represent TOPORS deletion constructs used in experiments presented in panel B. Diagram not to scale. <b>B)</b> Direct PPI between TOPORS, its deletion constructs and P26s4 tested by Y2H. Each experiment was performed six times; interactions recorded in a minimum of four out of the six experiments were interpreted as an overall positive result. The figure panel depicts a representative raw result from one of the six experiments. The bottom table includes a summary of results from all six experiments; total number of positive PPI results at a lower (D), or higher (Q) stringency level, are indicated for each tested PPI pair. Key: FL, full-length TOPORS (residues 1–1045); N, N-TOPORS; M, M-TOPORS; C, C-TOPORS. Positive PPI control (‘+’): AD-SV40 T Ag x BD-p53. Negative PPI control (‘–‘): AD-SV40 T Ag x BD-Lamin C; BD, GAL4 DNA <u>B</u>inding <u>D</u>omain; AD, GAL4 <u>A</u>ctivation <u>D</u>omain. D, medium selecting for X-α-galactose utilisation (blue colonies) and AbA resistance, i.e. activation of two PPI reporter genes; Q, medium selecting for X-α-galactose utilisation (blue colonies), AbA resistance, histidine synthesis and adenine synthesis, i.e. activation of four PPI reporter genes. <b>C)</b> P26s4 co-localised with all V5-tagged artificial TOPORS fragments at nuclei of hTERT-RPE1 cells, and it additionally co-localised with the V5-tagged C-TOPORS fragment in the cytoplasm of hTERT-RPE1 cells transfected with V5-tagged TOPORS deletion constructs, and co-stained for V5 and P26s4.</p

Ciliary-targeting sequences identified in P26s4 <i>in silico</i>.

<p>Ciliary-targeting sequences identified in P26s4 <i>in silico</i>.</p

A two-tier selection process led to identification of 21 putative interacting partners of TOPORS after the second selection level.

<p>A two-tier selection process led to identification of 21 putative interacting partners of TOPORS after the second selection level.</p

TOPORS, a Dual E3 Ubiquitin and Sumo1 Ligase, Interacts with 26 S Protease Regulatory Subunit 4, Encoded by the <i>PSMC1</i> Gene

<div><p>The significance of the ubiquitin-proteasome system (UPS) for protein degradation has been highlighted in the context of neurodegenerative diseases, including retinal dystrophies. TOPORS, a dual E3 ubiquitin and SUMO1 ligase, forms a component of the UPS and selected substrates for its enzymatic activities, such as DJ-1/PARK7 and APOBEC2, are important for neuronal as well as retinal homeostasis, respectively. TOPORS is ubiquitously expressed, yet its mutations are only known to result in autosomal dominant retinitis pigmentosa. We performed a yeast two-hybrid (Y2H) screen of a human retinal cDNA library in order to identify interacting protein partners of TOPORS from the retina, and thus begin delineating the putative disease mechanism(s) associated with the retina-specific phenotype resulting from mutations in TOPORS. The screen led to isolation of the 26 S protease regulatory subunit 4 (P26s4/ <i>PSMC1</i>), an ATPase indispensable for correct functioning of UPS-mediated proteostasis. The interaction between endogenous TOPORS and P26s4 proteins was validated by co-immuno-precipitation from mammalian cell extracts and further characterised by immunofluorescent co-localisation studies in cell lines and retinal sections. Findings from hTERT-RPE1 and 661W cells demonstrated that TOPORS and P26s4 co-localise at the centrosome in cultured cells. Immunofluorescent staining of mouse retinae revealed a strong P26s4 reactivity at the interface between retinal pigmented epithelium (RPE) layer and the photoreceptors outer segments (OS). This finding leads us to speculate that P26s4, along with TOPORS, may have a role(s) in RPE phagocytosis, in addition to contributing to the overall photoreceptor and retinal homeostasis via the UPS.</p></div

P26s4 co-localises with TOPORS and centrosomal markers in hTERT-RPE1 cells, but only at one centriole of the centrosome.

<p>P26s4 localised throughout the cell in a diffuse speckled pattern; the signal included distinct points co-localising with TOPORS staining (arrows). P26s4 co-localised with both PCM1 and PLK4 at one of the two centrioles; P26s4 signal is also observed at the linker molecules holding the two centrioles together. Scale bar: 10 μm; insets show a magnification of signals enclosed in the dashed squares. Fluorescent microscope images were taken using Zeiss Axiovert S100 inverted microscope. Images in the middle and bottom panel were deconvoluted, as described in Materials and Methods. Secondary antibody control images were collected using the same settings and generated no signals in the red and green channels.</p

Pull-down assays showing the interaction of His-tagged PRPF31 (WT and A216P mutant) with GST-tagged PRPF6 domains

Complexes between the two proteins were immobilized with glutathione sepharose beads and eluted with reduced glutathione. Panels show the Western analysis of elution products using immobilized GST-PRPF6N (amino acids 1–306), GST-PRPF6M (amino acids 307–607), and GST-PRPF6C (amino acids 607–941). Upper panels were probed with α-His.tag antibody and lower panels with α-GST antibody. Asterisks (*) indicate the size of full-length GST-PRPF6N, GST-PRPF6M, and GST-PRPF6C respectively. Note that the faint band in the upper panel for the pull-down assay with GST-PRPF6N is too small for PRPF31-His. Negative controls show absence of nonspecific binding of His-tagged PRPF31 to the beads and to GST tag. Quantification of the WT and mutant PRPF31 bands gave ratios of mutant:WT of 4.2±1.4 (for PRPF6M) and 2.1±0.5 (for PRPF6C). In each case, the mean and standard error was determined from four separate determinations.<p><b>Copyright information:</b></p><p>Taken from "Disease mechanism for retinitis pigmentosa (RP11) caused by missense mutations in the splicing factor gene "</p><p></p><p>Molecular Vision 2008;14():683-690.</p><p>Published online 18 Apr 2008</p><p>PMCID:PMC2324120.</p><p></p

Geographical origin of families with posterior polymorphous corneal dystrophy within the southwestern part of the Czech Republic.

<p>The geographical origin of the eldest members of each family is indicated by *. Twelve of the families, of which ten were genotyped and shown to share common haplotype, can be traced to a region of 13 km radius around the town of Klatovy. Two other genotyped families with the common full haplotype spanning over 23 Mb originate from an approximate 40 km radius from Klatovy, however knowledge of the place of origin only extended to three generations in both families.</p